Unlike the capillaries in such tissues as muscles and the like, the capillaries that supply the blood to most of the brain tissues except some areas including the circumventricular organs (pineal gland, pituitary body, area postrema, etc.) differ in that their endothelial cells are connected by tight intercellular junctions. Thus, passive transfer of substances from the blood to the brain is restricted, and although there are some exceptions, substances generally are unlikely to move into the brain from the capillaries except such compounds as are lipid-soluble or of low molecular weight (less than 200-500 Dalton) and electrically neutral around the physiological pH. This system, which restricts exchanges of substances between the blood and the tissue fluid of the brain through the endothelial cells of capillaries in the brain, is called blood-brain barrier or BBB. The blood-brain barrier restricts exchanges of substances not only between the blood and the brain but also between the blood and the tissue fluid of the central nervous system including the brain and the spine.
Owing to the blood-brain barrier, most of the cells of central nervous system escapes the effect of fluctuating concentrations of hormones and lymphokines in the blood, and thus are able to maintain their biochemical homeostasis.
The blood-brain barrier, however, imposes a problem when it comes to develop a medical drug. For example, although nerve growth factor (NGF), which is thought to be acting on cholinergic neurons in the central nervous system and working to maintain the viability of the cells by preventing apoptotic cell death, was expected to become a therapeutic drug for dementia caused by Alzheimer's disease, it was concluded that nerve growth factor would not function as a therapeutic drug for Alzheimer's disease because it, being unable to pass through the blood-brain barrier due to its molecular weight of over 10 kD, could not reach the affected site within the brain. Further, whereas an enzyme replacement therapy is carried out by intravenous supplementation with recombinant α-L-iduronidase as a therapy of mucopolysaccharide storage disease type 1 (Hurler syndrome), an inherited disease caused by α-L-iduronidase deficiency, the therapy is not effective for abnormality in the central nervous system (CNS) which is notable in Hurler syndrome because the enzyme cannot pass through the blood-brain barrier.
Development of various methods has been tried to enable the passage, through the blood-brain barrier, of such macromolecular compounds as proteins or the like necessary to be brought into function in the central nervous system. In the case of nerve growth factor, for example, attempts have been made for a method to cause the factor to pass through the blood-brain barrier by preparing it in a liposome-encapsulated form and letting those liposomes fuse with the cell membrane of endothelial cells in brain capillaries, but the attempts have failed to make the method materialize (Non-patent Document 1). In the case of α-L-iduronidase, an attempt was made to enhance the passive transfer of the enzyme through the blood-brain barrier by raising its blood concentration through an increased single dose of the enzyme, and it was demonstrated, using a Hurler syndrome animal model, that the abnormality in the central nervous system was ameliorated by the method (Non-patent Document 2).
Furthermore, an attempt has also been made to administer a macromolecular compound directly in the spinal cavity or into the brain. For example, reports have been made about a method in which human α-L-iduronidase was administered into the spinal cavity of a patient with a Hurler syndrome (mucopolysaccharide storage disease type 1)(Patent Document 1), a method in which human acid sphingomyelinase was administered into the brain ventricles of a patient with Niemann-Pick disease (Patent Document 2), and a method in which iduronate 2-sulfatase (I2S) was administered into the brain ventricles of Hunter syndrome model animals (Patent Document 3). While it seems possible by one of such methods to definitely let a medical drug act in the central nervous system, they are highly invasive.
There have been reported various methods to let a macromolecular compound get to the brain through the blood brain barrier, in which the macromolecular compound is modified to give it an affinity to a membrane protein occurring on the endothelial cells of brain capillaries, thereby inducing formation of its complex with the membrane protein, so that it then passes through the blood-brain barrier by endocytosis. Examples of those membrane proteins occurring on the endothelial cells of the brain capillaries include insulin, transferrin, insulin-like growth factor (IGF-I, IGF-II) as well as receptors for LDL and leptin.
For example, a technique has been reported in which nerve growth factor (NFG) is synthesized into the form of a fusion protein with insulin, and with the help of its binding to the insulin receptor, this fusion protein is allowed to pass through the blood-brain barrier (Patent Documents 4-6). Further, a technique has been reported in which nerve growth factor (NGF) is synthesized in the form of a fusion protein with anti-insulin receptor antibody, and with the help of its binding to the insulin receptor, this fusion protein is allowed to pass through the blood brain barrier (Patent Documents 4 and 7). Further, a technique has been reported in which nerve growth factor (NGF) is synthesized in the form of a fusion protein with transferrin, and with the help of its biding to transferrin receptor (TfR), this fusion protein is allowed to pass through the blood brain barrier (Patent Document 8). Further, a technique has been reported in which nerve growth factor (NGF) is synthesized in the form of a fusion protein with anti-transferrin receptor antibody (anti-TfR antibody), and with the help of its binding to transferrin receptor, this fusion protein is allowed to pass through the blood brain barrier (Patent Documents 4 and 9).
Looking further into the techniques that utilize anti-transferrin receptor antibody, there has been reported in the field of a technique to make a drug pass through the blood-brain barrier by biding it to an anti-TfR antibody, that a single-chain antibody can be used consisting of the heavy chain of an anti-TfR antibody on whose C-terminal side is bound, through a linker, its light chain (Non-patent Document 3). Further, an anti-hTfR antibody exhibiting a dissociation constant of 30 nM to 1 μM with hTfR can be used profitably in a technique to make a drug pass through the blood-brain barrier (Patent Document 10). Furthermore, it has been reported that a lysosomal enzyme such as I2S can be allowed to pass through the blood-brain barrier by preparing it into a fusion protein in which it is bound to an anti-TfR antibody (Patent Document 11). There also are reports of techniques based on an anti-hTfR antibody and liposomes in combination, in which a drug is led to pass through the blood-brain barrier by preparing it into an encapsulated form in liposomes that carry the anti-hTfR antibody on their surface (Patent Documents 12 and 13).
To consider utilization, as medicine, of a fusion protein constructed with the above antibody, there is a possibility that a hyper reaction like an immune response to the antibody could take place after the administration of the fusion protein, thus making its further administration difficult. Therefore, to prepare for such an event, it would be highly meaningful to provide fusion proteins in advance that are constructed with some antibodies different from those currently employed, in order to avoid termination of treatment because of such a hyper reaction.